Retinoic Acid Differentiation of HL-60 Cells Promotes Cytoskeletal Polarization

Experimental Cell Research 254, 130 –142 (2000) doi:10.1006/excr.1999.4727, available online at http://www.idealibrary.com on

Retinoic Acid Differentiation of HL-60 Cells Promotes Cytoskeletal Polarization Ada L. Olins,* Harald Herrmann,† Peter Lichter,‡ and Donald E. Olins* ,1 *Foundation for Blood Research, P.O. Box 190, 69 US Route One, Scarborough, Maine 04070-0190; and †Division of Cell Biology and ‡Organization of Complex Genomes, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg, Germany

Retinoic acid (RA) treatment of HL-60 cells in vitro induces granulocytic differentiation, involving reorganization of the nucleus and cytoplasm, development of chemoattractant-directed migration, and eventual apoptosis. The present studies with HL-60/S4 cells document that major elements of the cytoskeleton are changed: actin increases by 50%; vimentin decreases by more than 95%. The cellular content of a-tubulin does not significantly change; but the centrosomal– microtubule (MT) array moves away from the lobulating nucleus. Cytoskeletal-modifying chemicals modulate this polarized reorganization: Taxol and cytochalasin D enhance centrosome movement; nocodazole reverses it. Cytoskeletal-modifying chemicals do not appear to affect nuclear lobulation or the integrity of envelope-limited chromatin sheets (ELCS). Employing bcl-2-overexpressing HL-60 cells permitted demonstration of nuclear lobulation, ELCS formation, and centrosome–MT movement concomitantly during RA-induced differentiation, implying independence between the cellular reorganization and apoptotic programs. RA appears to promote an inherent potential in HL-60 cells for cytoskeletal polarization, likely to be important for chemoattractant-directed cell migration, an established characteristic of mature granulocytes. © 2000 Academic Press Key Words: centrosome microtubules; nuclear envelope; granulocytes; retinoic acid.

INTRODUCTION

In vitro chemoattractant-activated peripheral blood neutrophils acquire distinct cellular polarity, prior to and during locomotory activity [1–7]. This acquired polarity can be observed in terms of cell shape, cortical extensions, and cytoskeletal organization. To some extent, the exact cellular changes reflect the conditions employed in vitro. Neutrophils migrating on a glass slide along a linear chemoattractant gradient reveal 1 To whom reprint requests should be addressed. Fax: (203) 8831527. E-mail: dolins@ fbr.org.

0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

forward lamellipodia and a trailing uropod, both enriched in F-actin [4, 7]. Under these defined conditions, microtubules orient parallel to the direction of migration [2, 8] with the centrosomal region either closely associated to the lobulated nucleus [2, 6] or (in a minority of cells) positioned posterior to nucleus [2]. Also, under such conditions of directed chemotaxis, vimentin bundles accumulate within the uropod [9, 10]. Employing a different set of in vitro culture conditions (i.e., neutrophils attempting to migrate through a cell-impermeable/chemoattractant-permeable filter), the acquired cellular polarity reveals the centrosome anterior (i.e., closer to the filter) and the lobulated nucleus in a posterior position [1]. Neutrophils in blood or suspended in buffer are primarily spherical with the centrosomal region in a juxtanuclear position [4]. Acquired activated cellular polarity is established in only a few minutes following exposure to substrate and/or chemoattractant, implying that the differentiated granulocytic cell is “triggered” to convert rapidly from stationary to migratory form. The human myeloid leukemia cell line (HL-60) is widely investigated as a model for inducible cell differentiation [11–13]. HL-60 cells are capable of chemically induced differentiation into several paths following cessation of cell division: all-trans-retinoic acid (RA) and dimethyl sulfoxide (DMSO) stimulate granulocytic differentiation; 1,25-dihydroxyvitamin D 3, sodium butyrate, and 12-O-tetradecanoylphorbol-13-acetate (TPA) promote monocytic/macrophage differentiation. RA– or DMSO– granulocyte-differentiated HL-60 cells exhibit similarities and differences with normal blood neutrophils [11–14]. In parallel with normal neutrophils, RA- or DMSO-differentiated HL-60 cells exhibit nuclear lobulation, nitroblue tetrazolium reduction by superoxide anions, enhanced expression of the cell surface antigen CD11b, and phagocytotic capability. Also, in parallel to normal neutrophils, granulocytic, as well as monocytic/macrophage-differentiated, HL-60 cells eventually die by apoptosis [15–17]. The differences observed between RA- or DMSO-induced granulocytes and normal neutrophils include a higher proportion of immature granulocytic nuclear morphol-

130

RETINOIC ACID PROMOTES CYTOSKELETAL POLARITY IN HL-60

ogies after RA or DMSO treatment, compared to peripheral blood neutrophils, and the inability of chemically induced granulocytes to sort certain granule proteins [18]. A number of studies have explored the cytoskeletal properties of HL-60 cells, when induced to undergo granulocytic differentiation. Chemically induced myeloid differentiation leads to an increase (1.8- to 2.0fold) in total cell actin (including both G- and F-actin), with parallel increases in actin-binding proteins (i.e., myosin and gelsolin) [19, 20]. Exposure of myeloiddifferentiated HL-60 cells to the chemoattractant fMet-Leu-Phe (fMLP) promotes actin polymerization with a concomitant increase in cell cortex stiffness [21, 22]. Both undifferentiated and myeloid-differentiated HL-60 reveal locomotory behavior. However, the induced cells migrate faster (comparable in rate to normal neutrophils) and exhibit chemotactic responsiveness [21, 23]. fMLP-mediated chemotaxis is not detectable with undifferentiated HL-60 [23]. Myeloid differentiation of HL-60 cells leads to an apparent increase in microtubules [24, 25]. The functional significance of increased microtubules to the granulocytic state is unknown. Most of the mechanical properties of neutrophils are ascribed to actin and associated proteins [26]. Changes in the content of the intermediate filament protein vimentin during myeloid differentiation of HL-60 cells have been somewhat conjectural [see 27, for a summary of earlier studies]. The present study details the quantitative changes in cytoskeletal proteins and the cytoskeletal polarization observed during RA-induced differentiation of HL-60 cells. MATERIALS AND METHODS Cells. The HL-60 cell sublines employed in this study were described previously [27]. Most experiments used the rapidly differentiating cell line (HL-60/S4) [25], generously provided by Dr. A. C. Sartorelli (Yale University, School of Medicine, New Haven, CT). RA treatment of HL-60/S4 cells reaches peak granulocytic differentiation in about 4 days [25, 28], after which apoptosis begins to predominate. The Bcl2-overexpressing HL-60 cell subline HL-60 – bcl-2 [29] (kindly provided by Dr. S. J. Collins, Fred Hutchinson Cancer Research Center, Seattle, WA) was also examined to distinguish differentiation from apoptosis. All cell lines were cultivated in RPMI 1640 medium containing 10% fetal calf serum, with penicillin/streptomycin and glutamine added. HL-60 – bcl-2 cell cultures were maintained in the presence of 500 mg/ml G418, except during RA-induced cell differentiation, when G418 was absent. All cultures were grown at 37°C in a humid incubator purged with 5% CO 2/95% air. Chemicals. RA, TPA, Taxol, nocodazole, cytochalasin D, and poly-L-lysine (P-6516) were purchased from Sigma Chemical Company (St. Louis, MO). RA was dissolved in ethanol at a concentration of 1 mM; TPA was dissolved in acetone at a concentration of 160 mM. Both stocks were stored at 220°C. For differentiation, RA was employed at 1 mM; TPA, at 16 nmol/L. Taxol and nocodazole were dissolved as 10 mM stock solutions in DMSO; cytochalasin D was dissolved in DMSO at 5 mg/ml. These DMSO stock solutions were also stored at 220°C. In all experiments involving Taxol, nocodazole, or cytochalasin D, equivalent amounts of the DMSO solvent were

131

added to control cultures. No effects of the solvent alone could be detected. G418 was obtained from Gibco BRL (Gaithersburg, MD). Cell-Tak cell adhesive was obtained from Becton Dickinson Labware (Bedford, MA). Antibodies. The following mouse monoclonal antibodies were purchased from Sigma Immunochemicals (St. Louis, MO): anti-atubulin (T-5168); anti-tyrosine tubulin (T-9028); anti-acetylated tubulin (T-6793); anti-g-tubulin (T-6557); and anti-b-actin (A-5441). Mouse monoclonal anti-vimentin (clone Vim 3B4), used earlier [27], was from Progen (Heidelberg, Germany). Affinity-purified goat antihuman lamin B was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Affinity-purified rabbit anti-lamin B receptor (LBR) has been described previously [27]. Cy3-conjugated AffiniPure donkey anti-mouse IgG and FITC-conjugated AffiniPure donkey anti-goat IgG were produced by Jackson ImmunoResearch Laboratory, Inc. (West Grove, PA) and obtained from Dianova (Hamburg, Germany). Immunofluorescence. For most experiments, suspension cultures of HL-60/S4 cells (undifferentiated or RA-treated for 4 days) were counted in a hemocytometer and diluted with PBS and 7.4% HCHO/ PBS to yield 10 5 cells/ml in 3.7% HCHO. Fixation of the suspended cells proceeded for 15 min at RT. Cells were centrifuged onto polyL-lysine-coated ethanol-cleaned SuperFrost glass slides using a Shandon Cytospin 3 (Life Sciences International Ltd., Cheshire, UK); 0.5 ml of cells was centrifuged for 4 min at 1000 RPM to yield ca. 5 3 10 4 cells/slide. Cell-Tak-coated slides were also employed, providing a somewhat higher yield of adherent cells in the immunostaining procedure. Slides were washed in 50 mM NH 4Cl for 1 min and two times with PBS for 1 min each, permeabilized with 0.1% Triton X-100 in PBS for 3 min, and washed extensively with PBS prior to application of antibodies. All monoclonal and polyclonal antibodies were diluted with PBS to recommended concentrations. Incubation with both primary and secondary antibodies was for 1 h at 37°C in a moist chamber under a glass coverslip. Slides were extensively washed with PBS, stained with DAPI, and mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA). Confocal images were collected on a Leica TCS SP spectral microscope (Leica Microsystems Heidelberg GmbH, Heidelberg, Germany), with excitation and emission conditions chosen to clearly resolve Cy3- and FITC-labeled secondary antibodies. Nonconfocal images were collected on a Zeiss axioplan microscope using a Photometrics Quantix CCD camera controlled by IPLab Spectrum 3.1.1 software. Quantitation of the shape of the centrosomal–MT complex were made on HL-60 cells that were fixed with HCHO prior to cytocentrifugation, permeabilizing, and immunostaining with anti-a-tubulin. The number of cells counted ranged from 130 to 180 for most experiments. The position of the centrosome relative to the nucleus was judged in a similar number of cells after staining with anti-gtubulin; the nuclear envelope could be clearly visualized with antilamin B staining. Nuclear shape was determined from the DAPI staining. From the cell counts in each category, the percentage of cells and 95% confidence limits were calculated. Immunoblotting. Cell extracts were prepared according to a scheme previously described [30]. Cells were washed in PBS containing the protease inhibitors Complete, Mini (Boehringer Mannheim GmbH, Mannheim, Germany), centrifuged, and suspended in a solution containing 0.53 PBS, 25 mM Mops, 1.0 mM EGTA, and 0.2% NP-40. After centrifugation, the supernatant was removed and diluted 2:1 with 33 Laemmli sample buffer and immediately boiled for 5 min. The pellet from centrifugation was placed on ice and incubated in 0.53 PBS, 25 mM Mops, 1.0 mM EGTA 1% NP-40, and 250 units/ml Benzonase (E. Merck, Darmstadt, Germany). After 3 min of pipetting up and down so that the solution was no longer viscous, 2:1 vol of 33 Laemmli sample buffer was added and the entire sample was boiled for 5 min. When necessary, an additional benzonase incubation at 37°C was performed. A 1:1 mixture of these two extracts (supernatant and pellet) was prepared, separately for undif-

132

OLINS ET AL.

ferentiated and for RA-differentiated cells; the extracts contained no precipitates. Each gel lane contained the extract from the same number of cells of undifferentiated or RA-treated cells. SDS–polyacrylamide gels (10%) were run in Laemmli buffer at 25 mA and then electroblotted onto PVDF membranes (Millipore, Bedford, MA) with 25 mM sodium borate, 1 mM EDTA, pH 9.2 [31]. The current was raised by 100 mA every 5 min and continued at 500 mA for 1 h. Membranes were stained with Ponceau S (Sigma Immunochemicals), dried, and stored overnight. Immune reactions were carried out in 10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20 (TBST) with 5% dried milk at RT. Membranes were blocked for 1 h, incubated with primary antibody for 1 h, washed three times for 10 min with TBST, incubated with peroxidase-coupled goat anti-mouse IgG and IgM (Jackson ImmunoResearch Laboratory, Inc.) for 1 h, washed as above, and developed with enhanced chemiluminescent blotting detection reagents (Amersham, Pharmacia, Biotech, Piscataway, NJ) solutions for 1 min; various time exposures were collected on film. For quantitative determinations, the same gel contained numerous samples with known amounts of protein and the cell extracts. Tubulin (TL238) and non-muscle actin (APH99) both from cytoskeleton (TEBU GmbH, Frankfurt am Main, Germany) and recombinant human vimentin [32] were used as standards. Software used to quantify the intensity of digitized gel bands is Bio Image Intelligent Quantifier. Standard curves and concentrations of proteins in cell extracts were calculated using Kaleidagraph (Synergy Software, Reading, PA).

RESULTS

Retinoic Acid Induces Changes in the Amounts of Major Cytoskeletal Proteins Previous studies have indicated that chemically induced granulocytic differentiation of HL-60 cells results in an approximate 50% decrease in total cellular protein [19] and a 35% decrease in cell volume [33]. In addition, an earlier study [25] of RA-induced granulocytic differentiation in HL-60/S4 indicated that the amounts of tubulin and vimentin increased by day 4 of treatment. These measurements were obtained on fixed and permeabilized cells, stained with fluorescent antibody, and analyzed by flow cytometry. Since we observed [27] a dramatic decrease in total cellular vimentin by immunoblotting of RA-treated HL-60/S4

FIG. 1. Immunoblots of anti-actin, anti-a-tubulin, and anti-vimentin against total extracts of HL-60/S4 cells. O, undifferentiated cells; RA, granulocyte-differentiated cells after 4 days of retinoic acid treatment. Extracts of equal numbers of cells were loaded for each pair of lanes. Only the portion of the lanes which showed an ECL signal are shown. A small amount of proteolytic breakdown is observed in the reaction with vimentin. In each case the mobility of the bands shown was identical to the mobility of purified actin, tubulin, and vimentin, respectively, which were run as concentration standards on the same gel.

TABLE 1 Protein Content /Cell 0

RA

RA/0

Protein

Content

6SE

Content

6SE

Ratio

6SE

Actin a-Tubulin Vimentin

3.63 pg 1.64 pg 8.21 fg

0.36 0.13 0.84

5.23 pg 1.56 pg ,0.08 a

0.55 0.11 —

1.46 0.96 ,0.01

0.07 0.03 —

a

Intensity of band was ,1% of undifferentiated cell extract.

cells (4 days), a rigorous quantitation by immunoblot analysis of several major cytoskeletal proteins was undertaken. For this purpose, purified b-actin, a-tubulin, and vimentin were employed to construct immunoblot standard curves (data not shown). Undifferentiated and RA-treated (4 days) HL-60/S4 cell stocks were prepared, counted, incubated with protease inhibitors, and extracted into SDS sample buffer. During immunoblot comparisons of undifferentiated and RA-treated cells, adjacent lanes contained protein amounts corresponding to identical numbers of cells. Several examples of immunoblots are presented in Fig. 1. A summary of the quantitative determinations, expressed as picograms (or femtograms) of protein per cell are shown in Table 1. This quantitative immunoblotting analysis revealed that: (i) b-actin content increased by about 50%, somewhat less than previously reported [19, 20]; (ii) a-tubulin content remained essentially unchanged; and (iii) vimentin content of RA-treated cells disappeared nearly completely, to less than 1% of the level present in undifferentiated cells. The high molecular weight cytoskeletal cross-bridging molecule plectin [31, 34] was also examined by immunoblotting (data not shown). Analysis of band intensities of both undifferentiated and RA-treated HL-60/S4 cells indicated that this intermediate filament (IF)-binding protein is not down-regulated along with vimentin. These immunoblotting measurements document that significant changes are occurring in the total cell contents of major cytoskeletal proteins during the RA-induced differentiation process. Immunoblot analysis of the relative amount of LBR [35, 36] was also performed on the extracts of undifferentiated and RA-treated (4 days) HL-60/S4 cells. A significant increase (approximately three- to fourfold) of intact (58 kDa) band intensity was measured (Fig. 2), without the major appearance of the lower molecular weight cross-reactive bands observed previously [27]. Thus, a major increase in the total content of intact LBR is observed during RA-induced granulopoiesis (a previous analysis of lamin B [27] did not reveal any significant increase in relative amount), underscoring the question of whether this increase in LBR is

RETINOIC ACID PROMOTES CYTOSKELETAL POLARITY IN HL-60

FIG. 2. Immunoblot of anti-lamin B receptor (LBR) against total extracts of HL-60/S4 cells. O, undifferentiated cells; RA, granulocytedifferentiated cells after 4 days of retinoic acid treatment. Extracts of equal numbers of cells were loaded on the pair of lanes. Molecular weights of the markers in descending order are 158.2, 66.4, 55.6, 42.7, 36.5, and 26.6 kDa. Long exposures of the membrane to the film show a small amount of LBR cross-reactive polypeptides at 55 and 30 kDa.

causally related to the appearance of nuclear lobulation and formation of ELCS [27]. Retinoic Acid Promotes Reorganization of the Interphase Centrosomal–Microtubular Complex In view of the changes in cytoskeleton protein amounts and the documented changes in HL-60 cell behavior [21–23] following chemically induced granulocytic differentiation, immunofluorescent microscopy was employed to examine the corresponding changes in cytoskeletal organization. Studies were performed on cells that had been cytocentrifuged onto microscope slides after HCHO fixation in order to preserve 3-D structure. Following staining for a-tubulin and lamin B, confocal immunofluorescence microscopy was performed in order to visualize the 3-D arrangement of MTs and

133

their relationship to the nucleus in undifferentiated and RA-treated cells (Figs. 3A and 3B). The relationship of the centrosomal region to the cell nucleus was explored by combining staining with anti-g-tubulin and anti-lamin B (Figs. 3C and 3D). Figure 3A displays a typical undifferentiated interphase cell with the MT network (red) surrounding the nucleus (green) in an “ovoid” configuration. Figure 3B displays a representative RA-differentiated HL-60/S4 cell, with the MTs exhibiting a “slightly prolate” configuration. In this cell, the centrosomal region is “distal” from the indented nucleus and the MTs are aligned in a “parallel array” emanating from the centrosomal region. Brightly stained ELCS can be clearly identified between the nuclear lobes. Figures 3C and 3D display the relationships of the centrosomal region (red) to the nucleus (green): undifferentiated cells reveal the centrosomal region to be primarily “proximal” (i.e., close to) the nuclear envelope (Fig. 3C); the RA-differentiated cell reveals a more distal position of the centrosome (Fig. 3D). ELCS are clearly seen between nuclear lobes in this cell. Stereo confocal images permit an important understanding of the centrosomal–MTs structural reorganization; however, nonconfocal visual inspection (i.e., focusing up and down on individual cells) and documentation by CCD digitization was undertaken to provide quantitation of these distinguishable cytoskeletal states. A gallery of digitized nonconfocal images of undifferentiated and RA-treated HL-60/S4 cells, fixed with HCHO prior to cytocentrifugation and stained for a-tubulin or g-tubulin and DNA (DAPI), are presented in Fig. 4. Two examples of undifferentiated cells (Figs. 4A– 4D) reveal the anti-a-tubulin-stained proximal centrosomal region within the ovoid network of MTs. Figures 4E– 4H present two RA-treated cells stained with anti-a-tubulin: Figs. 4E and F are an example of a “prolate” cell; Figs. 4G and 4H, “slightly prolate.” Two undifferentiated cells stained with anti-g-tubulin are shown in Figs. 4I– 4L; both reveal the centrosome to be proximal to the ovoid nucleus. Figures 4M– 4P display two RA-treated cells with centrosomal regions distal from the lobulated nuclei. In undifferentiated and RAtreated cells, anti-g-tubulin staining also reveals diffuse cytoplasmic fluorescence attributable to the presence of cytosolic g-tubulin [37]. Three classes of structural parameters were investigated on all the immunostained preparations of cells: (i) shape of the MT network; (ii) centrosomal position; and (iii) nuclear shape. Compilations of many measurements for undifferentiated and RA-treated HL-60/S4 cells are presented as bar graphs in Figs. 4Q– 4S. These measurements clearly document that the two cell states (undifferentiated and RA-differentiated) are not homogeneous; but each has a characteristic cytoskeletal con-

134

OLINS ET AL.

RETINOIC ACID PROMOTES CYTOSKELETAL POLARITY IN HL-60

figuration and nuclear shape that describes the majority of cells. Cytoskeletal and Nuclear Responses to Treatments by Cytoskeleton-Modifying Chemicals The observation that a minority of undifferentiated HL-60/S4 cells exhibit a more differentiated cytoskeletal morphology (Fig. 4Q) suggests that undifferentiated cells are “triggered” to undergo structural reorganization. Likewise, finding a minority of RA-treated cells with a more immature morphology provokes questions about the stability of the cytoskeletal structural changes. These questions were explored by exposing undifferentiated and RA-treated HL-60/S4 cells to a variety of cytoskeleton-modifying chemicals: a MT-stabilizing drug (Taxol, 10 mM, 4 h); a MT-destabilizing drug (nocodazole, 10 mM, 4 h); and an actin filamentdestabilizing drug (cytochalasin D, 5 mg/ml, 2 h). After each drug treatment, cells were fixed to preserve 3-D structure and immunostained. Galleries of digitized images of immunostained cells are displayed in Fig. 5; bar graph compilations of measurements are presented in Fig. 6, allowing comparison with “control” undifferentiated and RA-treated cells (Fig. 4). Taxol treatment had a profound effect on both undifferentiated and RA-treated cells, essentially strengthening the cytoskeletal structural reorganization detailed earlier. Nuclear shape, however, was largely unaffected by Taxol treatment. In undifferentiated cells, almost 80% of cells exhibited slightly prolate or prolate MT arrays; but over 80% of cells retained ovoid nuclei. Centrosomes also revealed an increase in distal positioning (ca. 60%). RA-treated HL-60/S4 cells exposed to Taxol showed additional reduction in ovoid MT arrays, with a marked increase (ca. 80%) of prolate arrays. Surprisingly, a corresponding increase in the proportion of distal centrosomes was not observed, compared to control RA-treated cells. Taxol is known to provoke MT network nucleation from noncentrosomal sites [38]. The increased MT arrays and prolate shapes of many of the Taxol-treated RA-differentiated HL60/S4 cells may derive from these alternative nucleation sites. Nocodazole treatment yielded a very different consequence to the structure of undifferentiated and RAtreated HL-60/S4 cells from that observed with RA alone or with Taxol. MTs were not detected after staining with anti-a-tubulin. Instead, the cytoplasm revealed bright regions of stain. Centrosomes were much

135

more difficult to visualize with anti-g-tubulin, than in control cells (i.e., cells not treated with nocodazole). Those that could be visualized and scored revealed that over 90% of the centrosomes were positioned proximal to the nucleus. Thus, dissolution of the MT arrays in both undifferentiated and RA-treated cells correlated with moving the centrosome to a proximal (juxtanuclear) position. By DAPI staining, nuclear shape seemed more irregular in nocodazole-treated undifferentiated cells, compared to control undifferentiated cells. In RA-treated cells, subsequent treatment by nocodazole did not appear to reduce the vast majority of indented or lobulated nuclear shapes. Cytochalasin D treatment shifted both undifferentiated and RA-treated HL-60/S4 cells to cytoskeletal rearrangements intermediate between control cells and Taxol-treated cells. Thus, fragmentation of actin filaments promotes more prolate MT arrays, as assayed by anti-a-tubulin. The proportion of undifferentiated cells with distal centrosomes increased; no significant effect was seen with RA-treated cells. Somewhat surprising effects on nuclear shape were observed by treatment with cytochalasin D: there appeared to be an increase of indented forms in undifferentiated cells and an increase of ovoid shapes in RA-treated cells. The occurrence of ELCS in the nuclear envelope of HL-60/S4 cells, undifferentiated or RA-treated (4 days), exposed to Taxol, nocodazole, or cytochalasin D was determined by immunostaining with goat antilamin B. Figure 7 displays a gallery of images of immunostained HL-60/S4 cell nuclei. These data demonstrate that undifferentiated cells do not acquire ELCS after the (relatively) brief treatments with cytoskeleton-modifying chemicals. Similarly, the presence of RA-induced ELCS is refractory to treatment by Taxol, nocodazole, or cytochalasin D. Thus, ELCS are modifications of the nuclear envelope that are not induced or obliterated by brief perturbation of the MT or actin filament systems. Retinoic Acid Promotion of Cytoskeletal Reorganization Is Independent of Apoptosis HL-60/S4 cells induced to granulocytic differentiation by treatment with RA begins to undergo apoptosis by day 5 [27; see Fig. 1]. In order to explore any relationship between reorganization of the centrosomal–MT complex and subsequent apoptosis, HL-60 – bcl-2 cells [29] were incubated for 3 weeks with RA. At weekly intervals samples were fixed with HCHO, cyto-

FIG. 3. Stereoimmunofluorescent confocal microscopic images of undifferentiated (A, C) and RA-treated (B, D) HL-60/S4 cells, stained with anti-a-tubulin (A, B), anti-g-tubulin (C, D), and anti-lamin B (A–D). Anti-tubulins detected with Cy3 (red); anti-lamin with FITC (green). The centrosomal regions appear juxtanuclear (proximal) in (A, C) and appear distal in (B, D). MTs appear more parallel after RA treatment; compare (B) to (A). ELCS are readily apparent between nuclear lobes (B, D). The stereo angle for all pairs is 15°.

136

OLINS ET AL.

centrifuged, permeabilized, and immunostained with anti-a-tubulin, anti-g-tubulin, and anti-lamin B. Results are presented in Fig. 8 and summarized as follows: (i) Undifferentiated HL-60 – bcl-2 reveal about 95% of cells with ovoid MT arrays; (ii) RA-treated HL60 – bcl-2 cells reveal 50% slightly prolate-to-prolate MT arrays by 1 week, which remains fairly constant over the 3-week period of RA treatment; (iii) approximately 95% of centrosomal regions are proximal to the nucleus in undifferentiated cells; and (iv) about 75% of centrosomal regions remain proximal to the nucleus, even after 3 weeks of exposure to RA. Most nuclei appeared lobulated by 1 week of incubation with RA. Anti-lamin B-stained ELCS were seen routinely in lobulated nuclei by 1 week, in agreement with the ultrastructural data [27]. HL-60 – bcl-2 cells die slowly during the 3-week RA exposure period. Apparently, the cytoskeletal reorganization of HL-60 – bcl-2 cells in response to RA treatment occurs within 1 week and remains stable throughout the incubation period, whereas the proportion of cells with ELCS continues to increase with time [27]. To the extent that apoptotic cell death is suppressed in differentiating HL-60 – bcl-2 cells, it can be concluded that cytoskeletal reorganization is independent of apoptosis, similar to the independence of granulocytic differentiation [29, 39] and of ELCS [27]. DISCUSSION

FIG. 4. Immunofluorescent staining of undifferentiated and RAtreated HL-60/S4 cells with anti-a-tubulin and anti-g-tubulin. Two examples of undifferentiated cells stained with anti-a-tubulin are presented (A–D); two RA-treated cells stained with anti-a-tubulin (E–H). Undifferentiated cells stained with anti-g-tubulin (I–L); RAtreated cells stained with anti-g-tubulin (M–P) Anti-a-tubulin staining (A, C, E, G); anti-g-tubulin staining (I, K, M, O). DAPI staining (B, D, F, H, J, L, N, P). Nuclei lobulation can be observed (F, H, N, P). The centrosomal region appears proximal (juxtanuclear) in (A, C, I, K); distal in (E, G, M, O). All magnifications are the same; bar, 10 mm. Quantitation of the shape of MT arrays, centrosome position relative to the nucleus, and nuclear shape are presented in frames (Q–S), respectively. Measurements of undifferentiated HL-60/S4 cells (dark bars); RA-treated cells (light bars). Three different categories of MT array shapes detected by immunofluorescent staining with anti-a-tubulin were distinguished: O, ovoid; SP, slightly prolate; P, prolate. Two different categories of centrosome positions as detected by immunofluorescent staining with anti-g-tubulin were distinguished: P, proximal (juxtanuclear); D, distal. Three different

Granulopoiesis in response to growth factors and cytokines is a complex biological process involving myeloid-specific gene expression, leading to specific nuclear and cytoplasmic structural differentiation, changes in cellular behavior [1–10, 40 – 42], and eventual apoptosis [15]. Model cell systems, such as HL-60 [11–13], play an important role in analyzing the various mechanisms underlying these cellular differentiation events. In vitro granulopoiesis can be performed in a matter of days with sufficient numbers of cells for biochemical and microscopic analyses. The present study demonstrates that RA induces major cytoskeletal changes in granulocytic differentiating HL-60 cells, involving changes in the amounts of actin and vimentin and changes in the 3-D organization of the interphase centrosomal–MT complex. The 50% increase in actin content per cell, observed here, is somewhat less than previously reported [19, 20], possibly due to use of a different cell subline, different inducing chemicals, and/or different measuring proce-

categories of nuclear shape as detected by DAPI staining were distinguished: O, ovoid; I, indented; L, lobulated. The percentage of cells in each class is plotted; bars denote 95% confidence limits.

RETINOIC ACID PROMOTES CYTOSKELETAL POLARITY IN HL-60

137

FIG. 5. Immunofluorescent staining with anti-a-tubulin and anti-g-tubulin of undifferentiated and RA-treated HL-60/S4 cells, exposed to cytoskeleton-modifying chemicals (Taxol, nocodazole, cytochalasin D). For each chemical employed: one example of an undifferentiated cell stained with anti-a-tubulin is presented (A–C); one RA-treated cell stained with anti-a-tubulin (D–F); one undifferentiated cell stained with anti-g-tubulin (G–I); one RA-treated cell stained with anti-g-tubulin (J–L). Antibody staining is shown in (A, D, G, J); DAPI staining in (B, E, H, K); phase image in (C, F, I, L). All magnifications are the same; bar, 10 mm.

dures. However, the increase in actin content seems reasonable, in terms of the parallel development of locomotory behavior of myeloid-differentiated HL-60 cells [21–23]. The profound decrease (.95%) in vimentin content per cell is less easily interpretable in terms of cell behavior. Similar decreases in vimentin and disappearance of the IF network have been reported

previously in myeloid and erythroid differentiating systems in vitro [43, 44]. Immunofluorescent data on peripheral blood neutrophils [43, and unpublished observations by D. E. Olins] indicate that the remnants of the vimentin network are principally confined to perinuclear regions. Recent discussions on the probable role of IF networks to cell structure and function [45–

138

OLINS ET AL.

FIG. 7. Immunofluorescent staining with anti-lamin B of undifferentiated (A–D) and RA-differentiated (E–H) HL-60/S4 cells. Exposure to cytoskeleton-modifying drugs: (A, E) None; (B, F), Taxol; (C, G), nocodazole; (D, H), cytochalasin D. Notice the absence of ELCS in (A–D); their presence in (E–H). All magnifications are the same; bar, 10 mm.

FIG. 6. Quantitation of the shape of MT arrays, centrosome position relative to the nucleus, and nuclear shape for undifferentiated and RA-treated HL-60/S4 cells exposed to the cytoskeletonmodifying chemicals: Taxol (A–C), nocodazole (D), and cytochalasin D (E–G). Measurements of undifferentiated HL-60/S4 cells (dark bars); RA-treated cells (light bars). Three different categories of MT array shapes detected by immunofluorescent staining with anti-atubulin were distinguished: O, ovoid; SP, slightly prolate; P, prolate. Two different categories of centrosome positions as detected by immunofluorescent staining with anti-g-tubulin were distinguished: P, proximal (juxtanuclear); D, distal. Three different categories of nuclear shape as detected by DAPI staining were distinguished: O, ovoid; I, indented; L, lobulated. Following nocodazole treatment, MT arrays were not detected with anti-a-tubulin. Quantitative measurement of nuclear shape following nocodazole treatment was not performed; qualitative assessments are presented in the text. The percentage of cells in each class is plotted; bars denote 95% confidence limits.

47] emphasize that it is a dynamic system which may impart structural stability to the cytoplasm, to some extent by interactions with other major constituents of the cytoskeleton. In the case of the granulocytic differentiation of HL-60 cells, maintenance of an extensive IF network is clearly not a requirement for nuclear lobulation (and ELCS formation) or for reorganization of the centrosomal–MT complex. The cellular polarity acquired by RA-treated HL-60/S4 and HL-60 – bcl-2 cells was observed as a movement of the centrosomal–MT complex away from the nucleus, detected by immunostaining with anti-a- and anti-g-tubulin. This polarization positions the centrosome closer to the cell periphery and creates a more parallel array of MTs, which enclose the lobulated nucleus. A number of MT posttranslational modifications

have been described which correlate (but appear not to be causal) with changes in MT stabilization [48,49]: for example, (i) removal of the C-terminal tyrosine residue of a-tubulin (to generate a glutamic acid terminus) correlates with increased MT stability; and (ii) acetylation of a single lysine residue in a-tubulin correlates with increased MT stability. In view of the documented changes in MT stability observed during several morphogenetic events [48], RA-induced granulopoiesis was examined using mouse monoclonal anti-tyrosine tubulin or anti-acetylated tubulin antibodies (data not shown). Anti-tyrosine tubulin yielded strongly stained MT arrays in both undifferentiated and RA-treated cells, resembling those visualized with anti-a-tubulin. Anti-acetylated tubulin gave very weak staining with both undifferentiated and RA-treated cells. A few brightly stained MTs were seen in undifferentiated cells with anti-acetylated tubulin; none, in RA-differentiated cells. Immunoblot analyses indicated no changes in the band intensities of tyrosine tubulin, and a decrease in band intensities of acetylated tubulin, comparing RA-treated to undifferentiated HL-60/S4 cell extracts. Thus, in this system of induced granulocytic differentiation, immunostaining evidence sug-

FIG. 8. Kinetics of immunostaining patterns of HL-60 – bcl-2 cells incubated with RA for 3 weeks. Anti-a-tubulin staining (A): (—) ovoid shape of MTs around the nucleus; (- - -) slightly prolate; (z z z) prolate. Anti-g-tubulin staining (B): (—) proximal centrosomal position; (- - -) distal. The points represent the percentage of cells in each class; bars denote 95% confidence limits.

RETINOIC ACID PROMOTES CYTOSKELETAL POLARITY IN HL-60

gests that the formation of significant amounts of stabilized MTs does not take place. HL-60/S4 cells were also differentiated into the adherent macrophage/monocytic form by addition of TPA for 3 days, as described previously [27]. Such TPAdifferentiated cells do not acquire lobulated nuclei or ELCS. Employing the variety of anti-tubulin and antilamin B antibodies described above, the following consensus morphology was observed (data not shown): (i) nuclei were often irregular in shape, but without ELCS; (ii) MT arrays frequently radiated from a central aster, with the nucleus offset toward the cell periphery; (iii) many cells exhibited numerous brightly stained MTs reactive to anti-acetylated tubulin; and (iv) most cells exhibited moderate staining with antivimentin, frequently extending out into membrane processes. Overall, TPA-differentiated HL-60/S4 exhibited many differences from RA-differentiated cells in nuclear and cytoskeletal structure. Clues to the molecular mechanism of the polarization of the centrosome–MT complex in RA-treated HL-60 cells are presently quite fragmentary. From the present study, two observations will be focused upon: (i) the effects of Taxol and cytochalasin D on undifferentiated HL-60/S4 cells and (ii) the effects of nocodazole on RA-differentiated HL-60/S4 cells. Exposure of undifferentiated HL-60/S4 cells to relatively brief treatments of Taxol and cytochalasin D promote extension of the centrosome–MT complex to a more prolate configuration, without any obvious induction of nuclear lobulation or ELCS. Indeed, the effect of Taxol on acquired polarity is more profound than RA exposure for 4 days. To some extent this effect of Taxol is not surprising, since the rapid induction of parallel bundles of MTs after exposure to Taxol has been reported in both lymphocytes and neutrophils [8, 50 –53]. Unstimulated (or stimulated) lymphocytes exposed to Taxol (10 mM, 4 h) develop large bundles of MTs extending from a single centrosome, which is displaced toward the plasma membrane [50 –52]. Unstimulated peripheral blood neutrophils exposed to Taxol [53] or isolated from a patient infused with Taxol [8] revealed increased amounts of centrosome-associated MT bundles. The data from exposure to Taxol (present and published studies) suggest that movement of the centrosome toward the cell periphery is a consequence of MT polymerization and stabilization. The present observations that Taxol augments the polarization promoted in HL60/S4 cells by RA treatment suggests that MTs are formed and stabilized along the cell axis established by the centrosome–MT complex, even if noncentrosomal nucleation [38] is part of the Taxol effect. Interestingly, exposure of HL-60/S4 cells to cytochalasin D (5 mg/ml, 2 h) also mimics the effect of RA on centrosome–MT polarization in unstimulated cells and augments the RA effect in differentiated cells. A num-

139

ber of previous studies have implicated actin microfilaments in centrosome position and cell polarity. Neutrophils, incubated with TPA, spread on a substrate and exhibit centriole splitting and aster movement [54]. Nocodazole treatment prior to TPA prevents centriole splitting. Subsequent treatment with cytochalasin D leads to a rejoining of the split centrioles. The authors postulate that the spreading cell cortex (with the actin network) pulls the MTs, which in turn pull the centrosome apart. Disruption of the actin network eliminates this pulling force, allowing the MTs and centrioles to shift to a different “stable” state. A less dramatic effect is observed with neutrophils that have been induced to attempt migration through a cell-impermeable/chemoattractant-permeable filter [1]. As with control cells (i.e., not treated with cytochalasin B), the centrioles were proximal to (i.e., facing) the chemoattractant source; the nucleus, distal. By ultrastructural analysis, the centrosome-associated MTs of cytochalasin-treated cells appeared to be straighter than those of control cells, which appeared curved. In a different cell type (i.e., human lymphoblast KE 37) disruption of the actin microfilament system with cytochalasin D results in a dramatic cell elongation [55]. In this situation, the cell extensions depend upon intact MTs, since the induced polarity is suppressed by nocodazole and strengthened by Taxol treatment. These authors [55] suggest that an intact actin cortical network keeps the MTs bent, via actin–MT interactions. In yet another example, MDCK cells establish polarity after the formation of extensive cell junctions, resulting in the separation of centriolar pairs and their movement to the apical plasma membrane [56]. Cytochalasin D reverses this centriolar separation. The authors’ [56] interpretation is similar to [54]: (i) centrioles are pulled to the actin cortical network; and (ii) an opposing force, elongating MTs, pushes the centrioles back toward the cell center. Besides the role of actin in the locomotory behavior of neutrophils and HL-60 [4, 7, 21–23,26], there appears to be an additional role for actin, i.e., interacting with the centrosomal–MT complex and influencing cellular polarity [see 57, for discussion and further examples]. In the present study, nocodazole treatment of RAdifferentiated HL-60/S4 (as well as undifferentiated) cells resulted in a return of the centrosomal region to a juxtanuclear position. Unlike the models described above [54 –56], a role for intact MTs in moving the centrosomes back toward the nucleus can be eliminated. Also, since there is virtually no IF network, it can be discounted. Indeed, the mechanism for the frequently observed interphase nucleus– centrosome association [58] is still poorly understood. Early studies [59] indicated that this association is disrupted by pretreatment of interphase cells with cytochalasin B and nocodazole. In most interphase animal cells, the centrosomal region consists of paired centrioles sur-

140

OLINS ET AL.

rounded by a largely amorphous protein-rich matrix of pericentriolar material, some of which have been identified and characterized [60 – 63]. These proteins include g-tubulin and pericentrin (which together may be involved in MT nucleation), centrin, ninein, and other proteins [60 – 63]. Possibly, RA-differentiated, nocodazole-treated HL-60 cells will be a useful system to explore the factors facilitating interphase nucleus– centrosome association, since MTs are disrupted and IFs are virtually absent. In view of the well-established acquisition of cellular polarity by neutrophils, prior to and during locomotory behavior [1–7] and the evidence for locomotory behavior by both undifferentiated and myeloid-differentiated HL-60 cells [21, 23], it is likely that the functional significance of the cytoskeletal reorganization of HL-60 cells is related to the developing ability for cell migration. The establishment of a “cell axis” connecting nucleus and centrosome, parallel to the direction of migration has been described in other cell types (e.g., fibroblasts [64], African green monkey kidney cells during the wound response [65]; for other examples, exceptions and discussion, see [66, 67]). Undifferentiated HL-60/S4 cells appear to be “triggered” to undergo the cytoskeletal (and nuclear) reorganization. About 20 –25% of the undifferentiated cells exhibit movement of the centrosome away from a juxtanuclear position, with the establishment of a monopolar appearance of parallel MTs emanating from the centrosomal region; about 15% show indented or lobulated nuclei. All this occurs, even though the cells are growing in suspension, presumably not attached to a substrate nor exposed to any added chemoattractant. The cells do settle during the growth conditions, and fetal calf serum is present in the growth medium. Thus, it is not possible in the present studies to completely eliminate extracellular information as a factor to induce a basal level of cellular polarity and nuclear differentiation. Spinner flasks and serum-free defined media [12] may be useful to reduce the levels in undifferentiated cells. The present studies also do not permit any conclusion about whether the observed RA-induced movement of the centrosome is toward the “anterior” or “posterior” portion of the cell (defined by direction of migration). Early studies [68] employing MT inhibitors to study neutrophil locomotion concluded that functional MTs are essential for directed chemotaxis, but not for locomotion per se. In a summary of other data and a general discussion of mechanisms of chemotaxis [5], the authors point out that cells without functional centrioles or MTs can still migrate. However, such cells make more frequent and wider turns. Directed chemotaxis seems less well controlled than normal cells. The authors [5] develop a model for directed chemotaxis involving modulations of cell polarity (including MTs) which turn the cell toward the source of the chemoat-

tractant. These ideas have been further developed with the suggestion [49] that MTs may be critical to the spatial organization and coordination of signal transduction, assisting in the distribution of signaling molecules around the cytoplasm. The present studies with HL-60 – bcl-2 cells [29], and with cytoskeletal-modifying drugs, lead to an additional conclusion about the HL-60 cell system. It appears that induction of granulocytic differentiation by exposure to RA initiates at least three somewhat independent developmental programs: (i) cytoskeletal reorganization; (ii) nuclear lobulation and formation of ELCS; and (iii) apoptosis. The occurrence of both the RA-induced cytoskeletal and the nuclear changes in a bcl-2-overexpressing HL-60 cell subline, with prolonged survival due to suppressed apoptosis, supports this conclusion. The stability of nuclear lobulation and ELCS to treatment by Taxol, nocodazole, and cytochalasin D argues that these structures exhibit an integrity, independent from the integrity of the cytoskeleton. It will be of interest to explore the degree of independence between these major cellular events and other developmental programs (e.g., synthesis and packaging of granule proteins) during granulocytic differentiation. The authors express their appreciation to the following individuals at the German Cancer Research Center, Heidelberg, Germany: Lutz Edler, Renate Rausch, and Annette Kopp-Schneider, for assistance in statististical analyses; Monika Mauermann, for help with immunoblotting; and Dr. Jo¨rg Langowski, for advice on image processing. Dr. Werner Knebel (Leica Lasertechnik GmbH, Heidelberg) assisted in the collection of confocal immunofluorescent images. Dr. Brigitte Buendia (University of Paris VII) provided the affinity-purified antiLBR. A.L.O. and D.E.O. were supported by Guest Scientist fellowships from the German Cancer Research Center; H.H. was supported by the Fo¨rderprogramm der Gemeinsamen Forschungskommission der Medizinischen Fakulta¨t Heidelberg; and P.L. was supported by a grant (01KW9620) from BMBF “Foerderkonzept Humangenomforschung.”

REFERENCES 1.

2. 3.

4.

5.

6.

Malech, H. L., Root, R. K., and Gallin, J. I. (1977). Structural analysis of human neutrophil migration. J. Cell Biol. 75, 666 – 693. Zigmond, S. H., and Sullivan, S. J. (1979). Sensory adaptation of leukocytes to chemotactic peptides. J. Cell Biol. 82, 517–527. Anderson, D. C., Wible, L. J., Hughes, B. J., Smith, C. W., and Brinkley, B. R. (1982). Cytoplasmic microtubules in polymorphonuclear leukocytes: Effects of chemotactic stimulation and colchicine. Cell 31, 719 –729. Haston, W. S., and Wilkinson, P. C. (1988). Locomotion and chemotaxis of leukocytes: Gradient perception and locomotor capacity. Curr. Opin. Immunol. 1, 5–9. Devroetes, P. N., and Zigmond, S. H. (1988). Chemotaxis in eukaryotic cells: A focus on leukocytes and Dictyostelium. Annu. Rev. Cell Biol. 4, 649 – 686. Gudima, G. O., Vorobjev, I. A., and Chentsov, Y. S. (1988). Centriolar location during blood cell spreading and motion in vitro: An ultrastructural analysis. J. Cell Sci. 89, 225–241.

RETINOIC ACID PROMOTES CYTOSKELETAL POLARITY IN HL-60 7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Keller, H., Niggli, V., and Zimmermann, A. (1991). Diversity in motile responses of human neutrophil granulocytes: Functional meaning and cytoskeletal basis. Adv. Exp. Med. Biol. 297, 23– 37. Ding, M., Robinson, J. M., Behrens, B. C., and Vandre´, D. D. (1995). The microtubule cytoskeleton on human phagocytic leukocytes is a highly dynamic structure. Eur. J. Cell Biol. 66, 234 –245. Parysek, L. M., and Eckert, B. S. (1984). Vimentin filaments in spreading, randomly locomoting, and f-Met-Leu-Phe-treated neutrophils. Cell Tissue Res. 235, 557–581. Pryzwansky, K. B., and Merricks, E. P. (1998). Chemotactic peptide-induced changes of intermediate filament organization in neutrophils during granule secretion: role of cyclic guanosine monophosphate. Mol. Biol. Cell. 9, 2933–2947. Collins, S. J. (1987). The HL-60 promyelocytic leukemia cell line: Proliferation, differentiation, and cellular oncogene expression. Blood 70, 1233–1244. Breitman, T. R. (1990). Growth and differentiation of human myeloid leukemia cell line HL-60. In “Methods in Enzymology” (L. Packer, Ed.), Academic Press, New York. Yen, A. (1990). HL-60 cells as a model of growth control and differentiation: The significance of variant cells. Hematol. Rev. 4, 5– 46. Brackmann, D., Lund-Johanson, F., and Aarskog, D. (1995). Expression of cell surface antigens during the differentiation of HL-60 cells induced by 1,25-dihydroxyvitamin D3, retinoic acid and DMSO. Leukemia Res. 19, 57– 64. Savill, J. S., Wyllie, A. H., Henson, J. E., Walport, M. J., Henson, P. M., and Haslett, C. (1989). Macrophage phagocytosis of aging neutrophils in inflammation: Programmed cell death in the neutrophil leads to its recognition by macrophages. J. Clin. Invest. 83, 865– 875. Martin, S. J., Bradley, J. G., and Cotter, T. G. (1990). HL-60 cells induced to differentiate towards neutrophils subsequently die via apoptosis. Clin. Exp. Immunol. 79, 448 – 453. Solary, E., Bertrand, R., and Pommier, Y. (1994). Apoptosis of human leukemic HL-60 cells induced to differentiate by phorbol ester treatment. Leukemia 8, 792–797. Le Cabec, V., Calafat, J., and Borregaard, N. (1997). Sorting of the specific granule protein, NGAL, during granulocytic maturation of HL-60 cells. Blood 89, 2113–2121. Meyer, W. H., and Howard, T. H. (1983). Changes in actin content during induced myeloid maturation of human promyelocytes. Blood 62, 308 –314. Watts, R. G. (1995). Role of gelsolin in the formation and organization of triton-soluble F-actin during myeloid differentiation of HL-60 cells. Blood 85, 2212–2221. Meyer, W. H., and Howard, T. H. (1987). Actin polymerization and its relationship to locomotion and chemokinetic response in maturing human promyelocytic leukemia cells. Blood 70, 363– 367. Erzurum, S. C., Kus, M. L. Bohse, C. Elson, E. L., and Worthen, G. S. (1991). Mechanical properties of HL60 cells: role of stimulation and differentiation in retention in capillary-sized pores. Am. J. Respir. Cell Mol. Biol. 5, 230 –241. Verghese, M. W., Kneisler, T. B., and Boucheron, J. A. (1996). P 2U agonists induce chemotaxis and actin polymerization in human neutrophils and differentiated HL60 cells. J. Biol. Chem. 271, 15597–15601. Brown, W. J., Norwood, C. F., Smith, R. G., Snell, W. J. (1981). Development of capping ability during differentiation of HL-60 human promyelocytic leukemia cells. J. Cell. Physiol. 106, 127– 136.

141

25.

Leung, M.-F., Sokoloski, J. A., and Sartorelli, A. C. (1992). Changes in microtubules, microtubule-associated proteins, and intermediate filaments during the differentiation of HL-60 leukemia cells. Cancer Res. 52, 949 –954.

26.

Tsai, M. A., Waugh, R. E., and Keng, P. C. (1998). Passive mechanical behavior of human neutrophils: Effects of colchicine and paclitaxel. Biophys. J. 74, 3282–3291.

27.

Olins, A. L., Buendia, B., Herrmann, H., Lichter, P., and Olins, D. E. (1998). Retinoic acid induction of nuclear envelope-limited chromatin sheets in HL-60. Exp. Cell Res. 245, 91–104.

28.

Campbell, M. S., Lovell, M. A., and Gorbsky, G. J. (1995). Stability of nuclear segments in neutrophils and evidence against a role of microfilaments or microtubules in their genesis during differentiation of HL-60 myelocytes. J. Leukocyte Biol. 58, 659 – 666.

29.

Park, J. R., Robertson, K., Hickstein, D. D., Tsai, S., Hockenberry, D. M., and Collins, S. J. (1994). Dysregulated bcl-2 expression inhibits apoptosis but not differentiation of retinoic acid-induced HL-60 granulocytes. Blood 84, 440 – 445.

30.

Herrmann, H., and Wiche, G. (1983). Specific in situ phosphorylation of plectin in detergent-resistant cytoskeletons from cultured Chinese hamster ovary cells. J. Biol. Chem. 258, 14610 – 14618.

31.

Herrmann, H., and Wiche, G. (1987). Plectin and IFAP-300K are homologous proteins binding to microtubule-associated proteins 1 and 2 and to the 240-kilodalton subunit of spectrin. J. Biol. Chem. 262, 1320 –1325.

32.

Herrmann, H., Eckelt, A., Brettel, M., Grund, C., and Franke, W. W. (1993). Temperature-sensitive intermediate filament assembly. J. Mol. Biol. 234, 99 –113.

33.

Hallows, K. R., and Frank, R. S. (1992). Changes in mechanical properties with DMSO-induced differentiation of HL-60 cells. Biorheology 29, 295–309.

34.

Schro¨der, R., Warlo, I., Herrmann, H., van der Ven, P. F. M., Klasen, C., Blu¨mcke, I., Mundegar, R. R., Fu¨rst, D. O., Goebel, H. H., and Magin, T. M. (1999). Immunogold EM reveals a close association of plectin and the desmin cytoskeleton in human skeletal muscle. Eur. J. Cell Biol. 78, 288 –295.

35.

Ye, Q., Barton, R. M., and Worman, H. J. (1998). Nuclear lamin-binding proteins. In “Subcellular Biochemistry” (H. Herrmann and J. R. Harris, Eds.), Plenum, New York.

36.

Chu, A., Rassadi, R., and Stochaj, U. (1998). Velcro in the nuclear envelope: LBR and LAPs. FEBS Lett. 441, 165–169.

37.

Moudjou, M., Bordes, N., Paintrand, M., and Bornens, M. (1996). g-Tubulin in mammalian cells: The centrosomal and the cytosolic forms. J. Cell Sci. 109, 875– 887.

38.

Hyman, A., and Karsenti, E. (1998). The role of nucleation in patterning microtubule networks. J. Cell Sci. 111, 2077–2083.

39.

Naumovski, L., and Cleary, M. L., (1994). Bcl2 inhibits apoptosis associated with terminal differentiation of HL-60 myeloid leukemia cells. Blood 83, 2261–2267.

40.

Clarke, S., and Gordon, S. (1998). Myeloid-specific gene expression. J. Leukocyte Biol. 63, 153–168.

41.

Sanchez, J. A., and Wangh, L. J. (1999). New insights into the mechanism of nuclear segmentation in human neutrophils. J. Cell. Biochem. 73, 1–10.

42.

Berliner, N. (1998). Molecular biology of neutrophil differentiation. Curr. Opin. Hematol. 5, 49 –53.

43.

Dellagi, K., and Brouet, J.-C. (1984). Alterations of vimentin intermediate filaments expression during differentiation of HL60 and U937 human leukemic cell lines. Leukemia Res. 8, 611– 616.

142 44.

45.

46.

47. 48.

49. 50.

51.

52.

53.

54.

55.

56.

OLINS ET AL. Ngai, J., Capetanaki, Y. G., and Lazarides, E. (1984). Differentiation of murine erythroleukemic cells results in the rapid repression of vimentin gene expression. J. Cell Biol. 99, 306 – 314. Chou, Y.-H., Skalli, O., and Goldman, R. D. (1997). Intermediate filaments and cytoplasmic networking: New connections and more functions. Curr. Opin. Cell Biol. 9, 49 –53. Houseweart, M. K., and Cleveland, D. W. (1998). Intermediate filaments and their associated proteins: Multiple dynamic personalities. Curr. Opin. Cell Biol. 10, 93–101. Evans, R. M. (1998). Vimentin: The conundrum of the intermediate filament gene family. BioEssays 20, 79 – 86. Bulinski, J. C., and Gundersen, G. G. (1991). Stabilization of post-translational modification of microtubules during cellular morphogenesis. BioEssays 13, 285–293. Gundersen, G. G., and Cook, T. A. (1999). Microtubules and signal transduction. Curr. Opin. Cell Biol. 11, 81–94. Paatero, G. I. L., and Brown, D. L. (1982). Effects of taxol on microtubule organization and on capping of surface immunoglobulin in mouse splenic lymphocytes. Cell Biol. Int. Rep. 6, 1033–1040. Brown, D. L., Little, J. E., Chaly, N., Schweitzer, I., and PaulinLevasseur, M. (1985). Effects of Taxol on microtubular organization in mouse splenic lymphocytes and on response to mitogenic stimulation. Eur. J. Cell Biol. 37, 130 –139. Brown, D. L., Knox, J. D., and Paulin-Levasseur, M. (1992). The lymphocyte centrosome. In “The Centrosome” (V. I. Kalnins, Ed.), Academic Press, San Diego. Roberts, R. L., Nath, J., Friedman, M. M., and Gallin, J. I. (1982). Effects of Taxol on human neutrophils. J. Immunol. 129, 2134 –2141. Euteneuer, U., and Schliwa, M. (1985). Evidence for the involvement of actin in the positioning and motility of centrosomes. J. Cell Biol. 101, 96 –103. Bailly, E., Celati, C., and Bornens, M. (1991). The cortical actomyosin system of cytochalasin D-treated lymphoblasts. Exp. Cell Res. 196, 287–293. Buendia, B., Bre’, M.-H., Griffiths, G., and Karsenti, E. (1990). Cytoskeletal control of centrioles movement during the estab-

Received July 2, 1999 Revised version received September 30, 1999

lishment of polarity in Madin-Darby canine kidney cells. J. Cell Biol. 110, 1123–1135. 57.

Gavin, R. H. (1997). Microtubule–microfilament synergy in the cytoskeleton. Int. Rev. Cytol. 173, 207–242.

58.

Bornens, M. (1977). Is the centriole bound to the nuclear membrane? Nature 270, 80 – 82.

59.

Maro, B., and Bornens, M. (1980). The centriole-nucleus association: effects of cytochalasin B and nocodazole. Biol. Cell 39, 287–290.

60.

Bornens, M., and Moudjou, M. (1999). Studying the composition and function of centrosomes in vertebrates. Methods Cell Biol. 61, 13–34.

61.

Zimmerman, W., Sparks, C. A., and Doxsey, S. J. (1999). Amorphous no longer: the centrosome comes into focus. Curr. Opin. Cell Biol. 11, 122–128.

62.

Marshall, W. F., and Rosenbaum, J. L. (1999). Cell division: The renaissance of the centriole. Curr. Biol. 9, R218 –R220.

63.

Andersen, S. S. L. (1999). Molecular characteristics of the centrosome. Int. Rev. Cytol. 187, 51–109.

64.

Schutze, K., Maniotis, A., and Schliwa, M. (1991). The position of the microtubule-organizing center in directionally migrating fibroblasts depends on the nature of the substratum. Proc. Natl. Acad. Sci. USA 88, 8367– 8371.

65.

Euteneuer U., and Schliwa, M. (1992). Mechanism of centrosome positioning during the wound response in BSC-1 cells. J. Cell Biol. 116, 1157–1166.

66.

Kalnins, V. I., and Rogers, K. A. (1992). The centrosome in stationary and migrating endothelial cells. In “The Centrosome” (V. I. Kalnins, Ed.), Academic Press, San Diego.

67.

Schliwa, M., Euteneuer, U., Gra¨f, R., and Ueda, M. (1999). Centrosomes, microtubules and cell migration. Biochem. Soc. Symp. 65, 223–231.

68.

Bandmann, U., Rydgren, L., and Norberg, B. (1974). The difference between random movement and chemotaxis: Effects of antitubulins on neutrophil granulocyte locomotion. Exp. Cell Res. 88, 63–73.